The ability to elicit Abs with broad neutralization activity against HIV-1 has been a long sought-after but elusive goal of HIV/AIDS vaccine research. The long-term goal of this proposal is to understand how broad-spectrum neutralizing antibodies (BnAbs) are generated. The majority of the rare BnAbs (2F5, 4E10 and b12) isolated from HIV-1-infected individuals have the common characteristics of long IGHV-CDR3s with positively charged amino acids, structural features often seen in autoantibodies. The main hypothesis to be tested in this proposal is that natural induction of such autoreactive antibodies may be curtailed by the checkpoint control mechanisms occurring during B cell ontogeny. Therefore, a system that models a human immune system, is responsive to HIV infection/immunization and has demonstratable defects in B cell checkpoint control processes is an attractive platform to test this hypothesis. The humanized BLT and GTL mouse models are generated by transplantation of 3-irradiated NOD/scid and NOD/scid IL2r?-/- mice, respectively, with fetal liver and thymus tissues in addition to autologous CD34+ HSCs. The humanized mice allow efficient engraftment of multi-lineage human hemato-lymphoid cells, support a productive HIV-1 infection, develop Ab responses upon immunization with recombinant HIV gp140 trimer and demonstrate autoimmune properties. We will test the ability of these mice to generate strain-specific nAb and BnAbs against HIV-1 upon optimized immunization with gp140 Env trimers. Towards these goals, the specific aims of this proposal are: 1) To test several approaches including the addition of human cytokines and/or activated T cells to induce optimal antigen-dependent class Ig switching and a predominant IgG response in the HIVgp140 immunized BLT and GTL models. 2) To interrogate whether neutralizing antibodies are generated as a result of optimized immunization by HIV pseudotyped virus microneutralization assays. 3) To characterize and quantitate the quality of the neutralizing antibody (nAb) response by cloning and expressing anti-HIV Envelope (Env) Abs isolated by FACS sorting of gp140-trimer binding single B cells from B1-like, conventional B (B2) and Ab secreting cell (ASC)/plasma cell subpopulations. Sequence analysis and binding/neutralization studies of Abs derived from gp140-trimer binding B cell clones will provide a detailed molecular signature of BnAbs. Overall, our studies are aimed at developing a new small animal model to understand the long-standing question of how BnAbs are generated and to set the groundwork for vaccine studies that will result in generation of such BnAb responses. PUBLIC HEALTH RELEVANCE: A major reason for the failure of HIV vaccines is the lack of induction of broadly neutralizing anti-viral antibodies in the host. We propose to utilize new humanized mouse models to study the mechanism(s) by which BnAbs are produced and the barriers that must still be breached to elicit these types of antibodies by vaccination. The results of our studies will establish a new small animal HIV vaccine model and set the groundwork for vaccine studies aimed at the generation of potent and broadly neutralizing antibody responses to HIV.